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Although methanogens were recently discovered to occur in aerated soils, alpine regions have not been extensively studied for their presence so far. Here, the abundance of archaea and the methanogenic guilds Methanosarcinales, Methanococcales, Methanobacteriales, Methanomicrobiales and Methanocella spp. was studied at 16 coniferous forest and 14 grassland sites located at the montane and subalpine belts of the Northern Limestone Alps (calcareous) and the Austrian Central Alps (siliceous) using quantitative real-time PCR. Abundance of archaea, methanogens and the methanogenic potentials were significantly higher in grasslands than in forests. Furthermore, methanogenic potentials of calcareous soils were higher due to pH. Methanococcales, Methanomicrobiales and Methanocella spp. were detected in all collected samples, which indicates that they are autochthonous, while Methanobacteriales were absent from 4 out of 16 forest soils. Methanosarcinales were absent from 10 out of 16 forest soils and 2 out of 14 grassland soils. Nevertheless, together with Methanococcales they represented the majority of the 16S rRNA gene copies quantified from the grassland soils. Contrarily, forest soils were clearly dominated by Methanococcales. Our results indicate a higher diversity of methanogens in well-aerated soils than previously believed and that pH mainly influences their abundances and activities. Various methanogenic guilds were found to be present in aerated forest and grassland soils located at the montane and subalpine belts of the European Alps. Graphical Abstract Figure. Various methanogenic guilds were found to be present in aerated forest and grassland soils located at the montane and subalpine belts of the European Alps.

Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1), with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA) systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles.

The diversity of an archaeal community was analyzed in the water from a continental high-temperature, long-term water-flooded petroleum reservoir in Huabei Oilfield in China. The archaea were characterized by their 16S rRNA genes. An archaeal 16S rDNA clone library was constructed from the DNA isolated from the formation water, and 237 randomly selected positive clones were clustered in 28 phylotypes by sequencing analyses. Phylogenetic analysis of these sequences indicated that the dominant members of the archaeal phylotypes were affiliated with the order Methanomicrobiales. Totally, the archaeal community was composed of methanogens belonging to four orders: Methanobacteriales, Methanococcales, Methanomicrobiales, and Methanosarcinales. Most of the clones clustered with sequences previously described for methanogens, but there was a difference in the relative distribution of sequences detected here as compared to that of previous studies. Some thermophilic methanogens detected had been previously isolated from a number of high-temperature petroleum reservoirs worldwide; thus, they might exhibit adaptations to the environments and be the common habitants of geothermally heated subsurface environments.

Methyl coenzyme-M reductase A (mcrA) clone libraries were generated from microbial DNA extracted from the rumen of cattle fed a roughage diet with and without supplementation of the antimethanogenic compound bromochloromethane. Bromochloromethane reduced total methane emissions by c. 30%, with a resultant increase in propionate and branched chain fatty acids. The mcrA clone libraries revealed that Methanobrevibacter spp. were the dominant species identified. A decrease in the incidence of Methanobrevibacter spp. from the clone library generated from bromochloromethane treatment was observed. In addition, a more diverse methanogenic population with representatives from Methanococcales, Methanomicrobiales and Methanosacinales orders was observed for the bromochloromethane library. Sequence data generated from these libraries aided in the design of an mcrA-targeted quantitative PCR (qPCR) assay. The reduction in methane production by bromochloromethane was associated with an average decrease of 34% in the number of methanogenic Archaea when monitored with this qPCR assay. Dissociation curve analysis of mcrA amplicons showed a clear difference in melting temperatures for Methanobrevibacter spp. (80-82 °C) and all other methanongens (84-86 °C). A decrease in the intensity of the Methanobrevibacter spp. specific peak and an increase for the other peak in the bromochloromethane-treated animals corresponded with the changes within the clone libraries.

Subsurface microbial communities supported by geologically and abiologically derived hydrogen and carbon dioxide from the Earth's interior are of great interest, not only with regard to the nature of primitive life on Earth, but as potential analogs for extraterrestrial life. Here, for the first time, we present geochemical and microbiological evidence pointing to the existence of hyperthermophilic subsurface lithoautotrophic microbial ecosystem (HyperSLiME) dominated by hyperthermophilic methanogens beneath an active deep-sea hydrothermal field in the Central Indian Ridge. Geochemical and isotopic analyses of gaseous components in the hydrothermal fluids revealed heterogeneity of both concentration and carbon isotopic compositions of methane between the main hydrothermal vent (0.08 mM and -13.8 per thousand PDB, respectively) and the adjacent divergent vent site (0.2 mM and -18.5 per thousand PDB, respectively), representing potential subsurface microbial methanogenesis, at least in the divergent vent emitting more 13C-depleted methane. Extremely high abundance of magmatic energy sources such as hydrogen (2.5 mM) in the fluids also encourages a hydrogen-based, lithoautotrophic microbial activity. Both cultivation and cultivation-independent molecular analyses suggested the predominance of Methanococcales members in the superheated hydrothermal emissions and chimney interiors along with the other major microbial components of Thermococcales members. These results imply that a HyperSLiME, consisting of methanogens and fermenters, occurs in this tectonically active subsurface zone, strongly supporting the existence of hydrogen-driven subsurface microbial communities.

Methanogens and methanotrophs play unique roles as producers and consumers of the greenhouse gas methane (CH4) in soils, respectively. Here, we aimed to reveal whether and to which extent methane-cyclers occur in high-alpine soils, and to assess their spatial distribution along an altitudinal gradient (2700–3500 m) in the Austrian Alps at sites located within the alpine (2700–2900 m), the alpine-nival (3000–3100 m) and the nival belts (3200–3500 m). Methanococcales and Methanocella spp. were most abundant among all quantified methanogenic guilds, whereas Methanosarcinales were not detected in the studied soil. The detected methanogens seem to be capable of persisting despite a highly oxic low-temperature environment. Methanogenic and methanotrophic activities and abundances of methanotrophs, Methanococcales and Methanocella spp. declined with altitude. Methanogenic and methanotrophic abundances were best explained by mean annual soil temperature and dissolved organic carbon, respectively. Alpine belt soils harbored significantly more methane-cyclers than those of the nival belt, indicating some influence of plant cover. Our results show that methanogens are capable of persisting in high-alpine cold soils and might help to understand future changes of these environments caused by climate warming. Methanogenic archaea and methanotrophic bacteria were discovered in aerated high-alpine soils, indicating their ability to withstand low temperatures. Graphical Abstract Figure. Methanogenic archaea and methanotrophic bacteria were discovered in aerated high-alpine soils, indicating their ability to withstand low temperatures.

Methanogen populations of an intertidal mudflat in the Yangtze River estuary of China were investigated based on the methyl coenzyme M reductase A (mcrA) gene using 454-pyrosequencing and quantitative real-time polymerase chain reaction (qPCR). Samples were collected at six depths from three locations. In the qPCR analyses, a mean depth-wise change of mcrA gene abundance was observed from (1.23±0.13)×10 7 to (1.16±0.29)×10 8 per g dried soil, which was inversely correlated with the depletion of sulfate (R 2 =0.74; α=0.05) and salinity (R 2 =0.66; α=0.05). The copy numbers of mcrA was at least 1 order of magnitude higher than dissimilatory sulfate reductase B (dsrB) genes, likely indicating the importance of methanogenesis at the mudflat. Sequences related to the orders Methanomicrobiales, Methanosarcinales, Methanobacteriales, Methanococcales and the uncultured methanogens; Rice Cluster I (RC-I), Zoige cluster I (ZC-I) and anaerobic methane oxidizing archaeal lineage-1 (ANME-1) were detected. Methanomicrobiales and Methanosarcinales dominated the entire sediment layers, but detectable changes of proportions were observed with depth. The hydrogenotrophic methanogens Methanomicrobiales slightly increased with depth while Methanosarcinales showed the reverse. Chao1 and ACE richness estimators revealed higher diversity of methanogens near the surface (0—10 cm) when compared with the bottom sediments. The near-surface sediments were mainly dominated by the family Methanosarcinaceae (45 %), which has members that can utilize substrates that cannot be used by sulfate-reducing bacteria. Overall, current data indicate that Methanosarcinales and Methanomicrobiales are the most dominant methanogens within the entire depth profile down to 100 cm, with higher abundance and diversity of methanogens in the deeper and upper sediment layers, respectively.

In the present study nine promising primer sets, targeting Archaea and methanogenic Archaea in particular, were evaluated in silico, in vitro and in situ concerning specificity, accuracy and applicability in end-point (ep-) and especially quantitative (q-)PCR research. The main goal was to adapt and evaluate already adapted primer sets, which were partially designed in combination with TaqMan probes, in substantially cheaper SYBR Green-based qPCR applications. An initial 16S rRNA gene bank-based in silico evaluation revealed high coverage potentials for all primers within targeted groups, ranging from 71 to 90 %, except the Methanosaeta specific set showing a low potential of 37 %. Mentionable cross-reacting potentials could be detected for the Methanothermobacter, Methanomicrobiales and Methanoculleus sets. The in vitro evaluation with selected reference organisms revealed a specific behavior for most primer sets, while the Methanosarcina and Methanothermobacter sets showed most problematic cross-reactions in epPCR application. We were able to show that primers for detecting the total archaeal community, methanogenic orders Methanosarcinales, Methanobacteriales, Methanococcales and the genus Methanoculleus performed in a highly specific way and allowed an accurate quantification of targeted organisms without the use of expensive TaqMan probes. However, primer pairs designed for detecting Methanomicrobiales, Methanothermobacter, Methanosarcina and Methanosaeta are not suitable for SYBR Green applications. The reliability of in situ quantifications was assessed for a typical methanogenic community, derived from a thermophilic fermenter, and confirmed via denaturing gradient gel band quantification and sequencing. Thereby, we revealed high abundances of methanogenic Archaea, mainly comprising Methanoculleus and Methanosarcinales, while Methanobacteriales only formed a minor fraction.

The population structure of thermo- and mesophilic biogas reactors digesting maize silage as the sole substrate was investigated employing a novel, highly degenerated PCR-primer pair targeting mcrA/mrtA coding for the key enzyme of methanogens. No sequence affiliating with Methanococcales, Methanopyrales, ANME-, rice or fen soil clusters was detected. Direct MeA PCR-cloning results indicated that Methanobacteriales were the most important methanogens in the thermophilic reactors. 57% and 80% of the analysed sequences affiliated with this order, 14% and 20% with Methanosarcinaceae and 0% and 29% with Methanomicrobiales. Methanomicrobiales dominated in the mesophilic reactors at the given conditions, 69% and 84% of the sequences recovered from direct MeA primed cloning affiliated with this order, 31% and 0% with Methanosarcinaceae and 0% and 16% with Methanobacteriales. No sequence affiliating with Methanosaetaceae was found. MeA primed PCR-single-strand conformation polymorphism indicated that population fluctuations occurred. According to sequence analysis of excised bands, Methanosarcinaceae dominated and Methanobacteriales were significantly represented in the thermophilic fermenter. Only 1 Methanosaetaceae sequence was found. Hydrogenotrophs appear to have a much higher and obligate acetoclastic methanogens a much lower importance than previously thought in biogas production from renewable resources.

Sediments from Xuanwu Lake have been dredged in the past 3 years to improve the water quality, but methanogenesis should still exist in the newly settled sediment. Methane production, methanogens, and physiochemical parameters were detected in the surface sediments (0–5 cm) and/or vertical sediments (0–21 cm, segmented at interval of 3 cm). Methane flux at water–air interface varied among five detected sites. Principal component analysis showed that CH4 flux, content of water and the concentration of total nitrogen (TN), CH4 and organic matters (OM) weighed most heavily on the component I in surface sediments while different patterns were observed for vertical sediments. The copy number of the 16S rRNA gene for bacteria was lower in the surface sediment (0–6 cm) than that in deeper sediments (12–21 cm), while 16S rRNA genes of Archaea were almost evenly distributed in the vertical sediments. Representatives belonging to the orders Methanobacteriales, Methanomicrobiales, and Methanosarcinales were detected in all samples of the vertical sediments, except that no members of the Methanococcales were detected in the samples at 0–6 cm. The level of Methanobacteriales reached a highest density at 18.1 × 104 copies g−1 dry weight (dw) at 6–9 cm; for Methanosarcinales (76.89 × 106 copies g−1 dw) and Methanococcales (82.70 × 103 copies g−1 dw) at 12–15 cm, whereas for Methanomicrobiales (43.37 × 106 copies g−1 dw) at 9–12 cm. Methanosarcinaceae and Methanosaetaceae reached to their highest densities at 6–9 and 9–12 cm, respectively. These data provided useful information for better understanding the methanogenesis in the newly settled sediments of a recently dredged lake.